SUMMARY To date, millions of human genetic variants have been found, many in the coding or regulatory sequence of genes. However, for only a tiny fraction of these variants do we understand how the expression or function of the encoded product is affected. As a consequence, the promise of sequencing human genomes to understand human phenotypes ? especially the risk for many diseases with genetic components ? has gone largely unfulfilled. What is needed are facile, high-throughput methods for generating libraries of human cells bearing mutant sequence elements and for assessing these libraries to determine each variant's effect on molecular and cellular phenotypes. Thus, the Center for the Multiplexed Assessment of Phenotype, based largely in the University of Washington's Department of Genome Sciences, proposes to develop highly generalizable, reproducible and scalable technologies to generate, and assess the functional impact of, variants in human genes. In the first specific aim, the Center will establish two workhorse methods of mutagenesis to produce variants: saturation editing of genes at their endogenous loci in the human genome, and in vitro generation of variant libraries that are recombined into safe harbor sites. In the second specific aim, the Center will develop approaches to explore the impact of mutations in noncoding regions on versions of genes that have been minimized ? pared down to partially remove intronic sequence but still capable of providing essential activity. Further, it will develop mass spectrometry methods to analyze variation in coding sequences for its effect on protein abundance, stability, interactions, turnover and aggregation. In the third specific aim, the Center will assess variant effects on cell morphology, behavior and internal organization by using a novel, microscopy- based phenotyping technology, and on global transcription by developing a massively parallel single-cell mRNA profiling method. Center-developed technologies will be piloted on a set of human genes with disease relevance, enabling comparisons between each variant's functional effects and the effects of known pathogenic or benign variants. This effort will inform the use in the clinic of the large-scale functional data the Center's technologies will generate. Additionally, variants will be assessed under different conditions, such as in multiple cell lines, in combination with another mutation, or in the presence of a drug. The Center will also train early career experimentalists, clinical geneticists and data scientists to obtain and use large-scale functional data. This training will include internships in Center laboratories for one to three months, and apprenticeships for one to two years. These close interactions will generate medically- and biologically-relevant results and reveal the best paths for translating the vast amounts of Center-generated functional data for clinical use. Through these new technologies and their dissemination to the broader clinical community, the Center will advance the promise of the Human Genome Project by interpreting the vast landscape of human genetic variation.